Toxoplasma gondii, a member of the Apicomplexa, is a ubiquitous parasite of mammals. The predilection of this protozoan for the central nervous system causing necrotizing encephalitis constitutes its major presentation in patients with HIV infection. Because it can be transmitted by food or water T. gondii is classified as a category B NIH priority pathogen. During initial infection the rapidly proliferating tachyzoite stage of T. gondii differentiates into the slowly replicating bradyzoite stage that remain latent within tissue cysts for the life of the host; however reactivation of this latent infection can result in disease such chorioretintiis or encephalitis. Several lines of evidence suggest that bradyzoite differentiation is stress mediated and that the cyst wall (a modified parasitophorous vacuole membrane) contains many stage specific proteins and glycoproteins. In the present proposal, studies will be focused on the characterization of the components of the cyst wall. The components of the cyst wall will be characterized using a combination of proteomic, immunologic and genetic approaches taking advantage of techniques we have developed for purification of the T. gondii cyst wall. We have already identified several cyst wall specific proteins that have mucin type domains. Other investigators have previously noted that o-glycosylation occurs in T. gondii and is probably involved in differentiation. We will, therefore, also examine the role of o-glycosylation and mucin type linkages in the development of the cyst wall using biochemical and genetic approaches. Overall these studies will both elucidate the composition of the cyst wall and the glycoproteome of T. gondii providing reagents and insights that should prove useful in understanding the biology and organization of this critical structure. Understanding this structure is important for the development of new strategies to eliminate latent infection and prevent reactivation toxoplasmosis.